RESUMEN
Objective:To evaluate the effect of tropisetron on pyroptosis in cardiomyocytes subjected to hypoxia-reoxygenation (H/R) and the relationship with α7 nicotinic acetylcholine receptor (α7nAChR).Methods:Routinely cultured H9C2 cardiomyocytes were divided into 4 groups ( n=6 each) using a random number table method: control group (C group), H/R group, tropisetron plus H/R group (Tro+ H/R group), and α7nAchR antagonist MLA plus tropisetron plus H/R group (MLA+ Tro+ H/R group). H/R was produced by 12 h exposure of cells to hypoxia followed by 6 h reoxygenation in the other three groups except group C. Tropisetron at the final concentration of 10 nmol/L was added at 1 h before hypoxia in group Tro+ H/R.In group MLA+ Tro+ H/R, MLA was added at 2 h before hypoxia, and then 1 h later tropisetron at the final concentration of 10 nmol/L was given.At 6 h of reoxygenation, the pyroptosis rate of cardiomyocytes was determined by fluorescence immunostaining of caspase-1-AlexaFluor 488/DAPI, the concentrations of interleukin-1beta (IL-1β) and IL-18 in the supernatant were determined by enzyme-linked immunosorbent assay, the activity of LDH in the supernatant was measured by 2, 4 dinitrophenylhydra-zine colorimetric method, and the expression of α7nAchR, NLRP3 and caspase-1 in cardiomyocytes was detected by Western blot. Results:Compared with group C, the pyroptosis rate, activity of LDH and concentrations of IL-1β and IL-18 in the supernatant were significantly increased, the expression of NLRP3 and caspase-1 was up-regulated, and the expression of α7nAchR was down-regulated in group H/R ( P<0.05). Compared with group H/R, the pyroptosis rate, activity of LDH and concentrations of IL-1β and IL-18 in the supernatant were significantly decreased, the expression of NLRP3 and caspase-1 was down-regulated, and the expression of α7nAchR was up-regulated in group Tro+ H/R ( P<0.05). Compared with group Tro+ H/R, the pyroptosis rate, activity of LDH and concentrations of IL-1β and IL-18 in the supernatant were significantly increased, the expression of NLRP3 and caspase-1 was up-regulated, and the expression of α7nAchR was down-regulated in group MLA+ Tro+ H/R ( P<0.05). Conclusion:α7nAchR is involved in the process of tropisetron inhibiting pyroptosis in cardiomyocytes subjected to H/R.
RESUMEN
The voltage-gated Na channel subtype Nav1.7 is important for pain and itch in rodents and humans. We previously showed that a Nav1.7-targeting monoclonal antibody (SVmab) reduces Na currents and pain and itch responses in mice. Here, we investigated whether recombinant SVmab (rSVmab) binds to and blocks Nav1.7 similar to SVmab. ELISA tests revealed that SVmab was capable of binding to Nav1.7-expressing HEK293 cells, mouse DRG neurons, human nerve tissue, and the voltage-sensor domain II of Nav1.7. In contrast, rSVmab showed no or weak binding to Nav1.7 in these tests. Patch-clamp recordings showed that SVmab, but not rSVmab, markedly inhibited Na currents in Nav1.7-expressing HEK293 cells. Notably, electrical field stimulation increased the blocking activity of SVmab and rSVmab in Nav1.7-expressing HEK293 cells. SVmab was more effective than rSVmab in inhibiting paclitaxel-induced mechanical allodynia. SVmab also bound to human DRG neurons and inhibited their Na currents. Finally, potential reasons for the differential efficacy of SVmab and rSVmab and future directions are discussed.